Conjugated Linoleic Acid (CLA) is a conjugated diene isomer with the position and space of linoleic acid. 9c, 11t-CLA and 10t, 12c-CLA are two main active components. With the physiological mechanism that affects human health being gradually explained, CLA attracts more and more attention of the international food and pharmaceutical circles, and it has been determined that CLA is capable of combating cancer, preventing arteriosclerosis, losing weight, improving immunity, enhancing resistance of cells to free radicals and so on. Therefore, CLA has broad application prospect and huge market potential as a medicine, health food, functional food or food preservative, or in meat or feed industry.
It needs to prepare CLA by artificial means since there is almost no CLA in the nature. Natural triglyceride vegetable oil rich in linoleic acid is an important raw material for preparing CLA products. It is the best way to prepare high-purity and high-activity CLA through alkali-catalyzed conjugation and saponification with such vegetable oil.
At present, CLA is mainly produced with safflower oil (composition is shown in the table below) at home and abroad. This oil contains the most linoleic acid among all known plants. Its fatty acid composition is simple, containing about 5% and 3% of saturated C16:0 and C18:0 respectively, about 10% of unsaturated C18:1, up to 73-85% of linoleic acid, and almost no C18:3 and fatty acid with more carbon. Only the linoleic acid of C18:2 undergoes isomerization reaction and other fatty acids almost play no role during preparation of CLA through alkali isomerization. Therefore, CLA products can be directly prepared through conjugation with safflower oil featured by simple composition and low content of impurities.
Fatty acidContent (%)Fatty acid below C14 (<C14)<0.1Myristic acid (C14:0)<1.0Palmitic acid (C16:0)2-10Palmitoleic acid (C16:1)<0.5Stearic acid (C18:0)1-10Oleic acid (C18:1)7-12Linoleic acid (C18:2)55-81 Linolenic acid (C18:3)<0.1Arachidic acid (C20:0)<0.5Arachidonic acid (C20:1)<0.5Behenic acid (C22:0)<0.5
Relatively speaking, content of linoleic acid is low, and impurity composition is complicated in other vegetable oils, particularly content of some impurities similar to linoleic acid in property is relatively high, such as oleic acid. Oleic acid is similar to linoleic acid in structure and property. For example, their boiling point differs by less than 1° C., and their melting point and polarity are approximate. They are only different in number of double bonds. There are no effective methods for separation and purification, and rectification, low temperature crystallization, extraction, conventional adsorption chromatography, supercritical extraction and other common technologies can not realize effective separation, which makes it difficult to obtain CLA products of content over 80%. Therefore, it can not be used for preparation of CLA. Low yield and high price of safflower oil leads to the contradiction of insufficient raw materials for production of CLA and inefficient use of the abundant vegetable oils.
Silver ions can form π-complex with double bonds of unsaturated fatty acid, so they have specific binding force with double bonds. The number, position and configuration of double bonds have direct effect on the strength of binding force. Silver ions are loaded or bonded onto the adsorbent and separated because of difference in acting force of fatty acid with different number of double bonds and silver ions on the adsorbent. Silica gel is a common adsorbent, and silver-based silica gel can be obtained by adding silica gel into silver nitrate aqueous solution of certain concentration after mixing, suction filtration, drying and activation. Silver-based silica gel column chromatography is a method for step-by-step separation of fatty acid through a series of eluents with different polarities with silver-based silica gel as stationary phase, and a simple, timesaving, efficient and industrialized liquid chromatography. Research on separation of related substances with silver-loaded silica gel column has been reported at home and abroad.
A method for separation of linoleic acid in safflower oil with silver-based silica gel is introduced in patent CN 101921186A. High-purity linoleic acid products are obtained with safflower oil after silver-based silica gel column chromatography, but glyceride is used as raw material in the patent, which is bad for enrichment of linoleic acid and greatly reduces the recovery rate while obtaining high-purity products. A method for purification of EPA with silver-based silica gel is introduced in U.S. Pat. No. 0,027,865. EPA products of content over 90% can be obtained through silver-based silica gel column chromatography after two-step urea inclusion of fish oil fatty acid, but recovery rate is greatly reduced by the two-step urea inclusion.
J. L. Guil-Guerrero et al. studied the effect of separation and purification of silver-based silica gel chromatography for polyunsaturated fatty acid, and mainly separated and purified fatty acid with degree of unsaturation more than 2 with samples rich in polyunsaturated fatty acid; Jianxia Guo et al. conducted preliminary purification for linoleic acid with safflower oil through urea inclusion to remove saturated and monounsaturated fatty acids, and separated polyunsaturated fatty acid with silver-based silica gel column to obtain linoleic acid of purity more than 95%; Noorul Jannah Zainuddin et al. conducted silver-based silica gel chromatographic separation with catfish oil after preliminary urea inclusion and purification, and separated linoleic acid of purity over 90% from polyunsaturated fatty acid, but recovery rate of linoleic acid was only 12.9% due to influence of preliminary purification.
El Hassan et al. separated EPA products of content more than 90% from microalgae and fish oil with silver-based silica gel; Sajilata et al. separated γ-linolenic acid of content over 96% from spirulina with silver-loaded silica gel column.
It can be seen from the above patents and literatures that separation and purification of fatty acid with silver-loaded silica gel column mainly have the following deficiencies at home and abroad: separation of polyunsaturated fatty acids is dominant, such as linolenic acid, EPA, etc., and there is no special research report on separation of fatty acid of low saturation, particularly separation of oleic acid and linoleic acid; the samples are subject to urea inclusion before separation to remove saturated and monounsaturated fatty acids in the few purification technologies for linolenic acid, which finally becomes separation of polyunsaturated fatty acid; the raw material used in the report is safflower oil or fatty acid subject to preliminary purification that contains a relatively high proportion of linoleic acid, failing to clearly show the role of silver-loaded silica gel in purification of linoleic acid, and recovery rate of final samples is very low due to pre-treatment; the raw material is glyceride, which is bad for purification of linoleic acid due to existence of monoglyceride and diglyceride; meanwhile, the technologies reported basically are at the level of chromatographic analysis with a small sample size and complicated elution system, falling short of requirements of production. These factors restrict the application of this technology in industrialized production and preparation of linoleic acid.